This invention relates generally to fluorescent rhodamine dye compounds. More specifically, this invention relates to sulfonated diarylrhodamine dyes useful as fluorescent labeling reagents.
The non-radioactive detection of biological analytes utilizing fluorescent labels is an important technology in modern molecular biology. By eliminating the need for radioactive labels, safety is enhanced and the environmental impact and costs associated with reagent disposal is greatly reduced. Examples of methods utilizing such non-radioactive fluorescent detection include 4-color automated DNA sequencing, oligonucleotide hybridization methods, detection of polymerase-chain-reaction products, immunoassays, and the like.
In many applications it is advantageous to employ multiple spectrally distinguishable fluorescent labels in order to achieve independent detection of a plurality of spatially overlapping analytes, e.g., single-tube multiplex DNA probe assays and 4-color automated DNA sequencing methods. In the case of multiplex DNA probe assays, by employing spectrally distinguishable fluorescent labels, the number of reaction tubes may be reduced thereby simplifying experimental protocols and facilitating the production of application-specific reagent kits. In the case of 4-color automated DNA sequencing, multicolor fluorescent labeling allows for the analysis of multiple bases in a single lane thereby increasing throughput over single-color methods and reducing uncertainties associated with inter-lane electrophoretic mobility variations.
Assembling a set of multiple spectrally distinguishable fluorescent labels is problematic. Multi-color fluorescent detection imposes at least six severe constraints on the selection of dye labels, particularly for applications requiring a single excitation light source, an electrophoretic separation, and/or treatment with enzymes, e.g., automated DNA sequencing. First, it is difficult to find a set of structurally similar dyes whose emission spectra are spectrally resolved, since the typical emission band half-width for organic fluorescent dyes is about 40-80 nanometers (nm). Second, even if dyes with non-overlapping emission spectra are identified, the set may still not be suitable if the respective fluorescent quantum efficiencies are too low. Third, when several fluorescent dyes are used concurrently, simultaneous excitation becomes difficult because the absorption bands of the dyes are usually widely separated. Fourth, the charge, molecular size, and conformation of the dyes must not adversely affect the electrophoretic mobilities of the analyte. Fifth, the fluorescent dyes must be compatible with the chemistry used to create or manipulate the analyte, e.g., DNA synthesis solvents and reagents, buffers, polymerase enzymes, ligase enzymes, and the like. Sixth, the dye must have sufficient photostability to withstand laser excitation.
Currently available multiplex dye sets suitable in 4-color automated DNA sequencing applications require blue or blue-green laser light to adequately excite fluorescence emissions from all of the dyes making up the set, e.g., argon-ion lasers. Use of blue or blue-green lasers in commercial automated DNA sequencing systems is disadvantageous because of the high cost and limited lifetime of such lasers.
In a first aspect, the invention comprises sulfonated diarylrhodamine dye compounds having the structures: 
including nitrogen- and aryl-substituted forms thereof. At least one of R1, R3, R4, R5, R6, R8, R9, R10, R11, R13, R14, R15, R16, R17, R18, R19, R20, and R21 is sulfonate.
Nitrogen substituents, R2, R2xe2x80x2, R12 and R12xe2x80x2, may be C1-C6 alkylsulfonate or C4-C10 arylsulfonate. In certain embodiments, alkylsulfonate is xe2x80x94(CH2)nxe2x80x94SO3H, and n is an integer from 1 to 6, and arylsulfonate is: 
wherein n is 0 or 1.
In other embodiments, nitrogen substituents, R2, R2xe2x80x2, R12 and R12xe2x80x2, may be C1-C6 alkylcarboxylate or C4-C10 arylcarboxylate: 
wherein n is 0 or 1.
Another aspect of the invention includes energy-transfer dye compounds comprising a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response thereto; an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and a linker for linking the donor dye and the acceptor dye; wherein at least one of the donor dye and acceptor dye is a sulfonated diarylrhodamine compound.
Another aspect of the invention is a labelled nucleoside, nucleotide, polynucleotide or polypeptide wherein the label is a sulfonated diarylrhodamine compound or an energy-transfer dye comprising a sulfonated diarylrhodamine compound.
Another aspect of the invention is a labelling reagent, including phosphoramidite or active ester linking moieties of a sulfonated diarylrhodamine compound, which form covalent attachments with substrates and methods of labelling substrates with the reagents.
Another aspect of the invention is methods for forming a labelled substrate comprising the step of reacting a substrate with the linking moiety of a sulfonated diarylrhodamine compound or an energy-transfer dye comprising a sulfonated diarylrhodamine compound.
Another aspect of the invention is methods of generating a labelled primer extension product by extending a primer-target hybrid with an enzymatically-incorporatable nucleotide. The primer or the nucleotide may be labelled with a sulfonated diarylrhodamine compound or an energy-transfer dye comprising a sulfonated diarylrhodamine compound.
Another aspect of the invention is methods of polynucleotide sequencing by forming a mixture of four classes of polynucleotides where each class is labelled at the 3xe2x80x2 terminal nucleotide with a sulfonated diarylrhodamine compound or an energy-transfer dye comprising a sulfonated diarylrhodamine compound, and the labels are spectrally resolvable.
Another aspect of the invention is methods of oligonucleotide ligation by annealing two probes to a target sequence and forming a phosphodiester bond between the 5xe2x80x2 terminus of one probe and the 3xe2x80x2 terminus of the other probe wherein one or both probes are labelled with a sulfonated diarylrhodamine compound or an energy-transfer dye comprising a sulfonated diarylrhodamine compound.
Another aspect of the invention is methods of amplification by annealing two or more primers to a target polynucleotide and extending the primers by a polymerase and a mixture of enzymatically-extendable nucleotides wherein at least one of the primers or one of the nucleotides is labelled with a sulfonated diarylrhodamine compound or an energy-transfer dye comprising a sulfonated diarylrhodamine compound.
Another aspect of the invention is kits of reagents including a sulfonated diarylrhodamine compound or an energy-transfer dye comprising a sulfonated diarylrhodamine compound.
These and other features and advantages of the present invention will become better understood with reference to the following description, figures, and appended claims.